1. Field of the Invention
This invention relates to methods and devices for conducting a variety of assays for determining the presence of an analyte in samples using nucleotide conjugates.
2. Discussion of the Background Information
The detection of some target analytes in low concentrations is important for the early diagnosis of some illnesses. For example, recent research suggests that measuring cardiac Troponin I (cTnI) at levels below those of typical commercial assays provides information of value to clinicians. Note that even very low levels of cTnI, i.e., below those considered indicative of acute myocardial infarction, may be indicative of future adverse cardiovascular status. For example, Zethelius et al., (2006, “Troponin I as a predictor of Coronary Heart Disease and Mortality in 70-year-old men: A community-based cohort study”, Circulation, vol. 113:1071-1078) performed a study to investigate the correlation between cardiac TnI concentrations and the prediction of future coronary heart disease. They observed that in 70 year old men that showed no clinical signs of cardiovascular disease, but exhibited slightly elevated levels of cardiac TnI could predict a forthcoming coronary heart disease event. Furthermore, Apple et al., (2008, “Use of the Centaur TnI-Ultra Assay for Detection of Myocardial Infarction and Adverse Events in Patients Presenting With Symptoms Suggestive of Acute Coronary Syndrome”, Clinical Chemistry, vol. 54(4):723-728) performed a study in a sensitive troponin assay to assess the prognostic value of assessing the risk of short-term adverse events based on cTnI values at the limit of detection and 99th percentile reference value. They concluded that “our data add to the growing evidence that with improved, analytically robust cTn assays with low LoDs [levels of detection], any measurable cTnI implies a higher risk than cTnI concentrations below an assay's Loa.”
Current point-of-care immunoassay technology, although highly valuable in its ability to measure the presence and concentration of various target analytes, is somewhat limited in its ability to reliably detect very low levels of target analytes such as cTnI. Thus, the need exists for reliably detecting low levels of target analytes such as cTnI, particularly in point-of-care immunoassay analyte detecting devices.
U.S. Pat. No. 7,419,821, the entirety of which is incorporated herein by reference, utilizes a sandwich assay where two capture antibodies are immobilized on an electrode and two signal antibodies, e.g., FAB (Fragment Antigen Binding) antibody fragments, are labeled with an signal enzyme, such as alkaline phosphatase (ALP), to form a signal conjugate. An analyte, e.g., antigen such as cTnI, binds to the capture antibodies and the signal antibodies to form a sandwich assay, which provides a signal indicating the presence of the analyte. Conventionally, signal antibodies are bound to signal enzymes to form the signal conjugates through cross-linking technologies. These synthesis conditions lead to a wide range in the ratio of signal antibodies to label enzymes as well as in the number of signal enzymes per signal conjugate and in the number of signal antibodies per conjugate. For FAB antibody fragments, for example, there typically is a statistical population of signal conjugates having from no (0) FAB to an estimated 15 FAB per signal conjugate. As a result, the synthesized signal conjugates are typically purified by size exclusion chromatography to create a signal conjugate population having a narrower range of signal antibodies, e.g., FAB molecules, per signal conjugate. Such purification techniques, however, undesirably lead to reduced predictability and variable ratios of (i) signal antibodies to signal enzyme (e.g., FAB to ALP); (ii) signal enzyme to the signal conjugate, and (iii) signal antibody to signal conjugate. These variable ratios limit the ability of sandwich assays to reliably detect very low levels of target analytes in samples.
Enzyme-Linked Immunosorbent Assay (ELISA) based assays, used to assess the concentration of an analyte, e.g., antigen molecule in samples, including bodily fluids and environmental samples, conventionally require the ability to covalently link a signal antibody to an signal enzyme to generate the detection component of the assay. A history and review of this technology can be found in Lequin (2005, Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA), Clinical Chemistry, vol 51(12): 2415-2418). The need exists for increasing the sensitivity of ELISA assays as some antigens are present at extremely low concentrations.
U.S. Pat. No. 5,164,311 discloses an antibody-enzyme conjugate produced by adding sulfhydryl groups to an antibody and maleimidyl groups to an enzyme to produce a modified antibody and enzyme, and reacting the modified antibody and enzyme to produce the conjugate. In doing so, the '311 patent states that: “It would be advantageous to provide a labeling system wherein direct antibody-enzyme conjugates could achieve high enzyme-to-antibody ratios and therefore provide a higher degree of sensitivity approaching or equal to the avidin-biotin labeling system. Such an improved direct antibody-enzyme conjugate would not require the additional incubation step and washing steps as the biotin-avidin labeling system requires.” The '311 patent describes the use of cross-linking agents such as SPDP with crosslinking molecules with the ability to crosslink sulfhydryl and maleimide groups for enzyme-antibody conjugates, however it is silent on using this for synthetic oligonucleotides.
Synthesis of conjugates based on enzymes and synthetic oligonucleotides have been developed for the molecular biology application of DNA hybridization techniques like Southern hybridization (Reyes & Cockerell, 1993, “Preparation of pure oligonucleotide-alkaline phosphatase conjugates”, Nucleic Acids Research, vol 21(23): 5532-5533). The application of these conjugates does not anticipate their use for ELISA based assays.
With the development of an extremely sensitive DNA amplification strategy, e.g. Polymerase Chain Reaction (PCR) covered by U.S. Pat. No. 5,656,493 and others, it has been recognized that the combination of ELISA and PCR could increase detection sensitivity. For example, Sano et al., (2000, “Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates, Science, vol. 258(5079): 120-122) describe one of the earliest applications of this strategy to antigen detection. There are many references to the application of this approach including Kozlov et al., (2004, “Efficient strategies for the conjugation of oligonucleotides to Antibodies Enabling Highly Sensitive Protein Detection”, Biopolymers, vol 73: 621-630) who describe synthetic oligonucleotide antibody conjugation synthesis strategies along with application of these specific conjugates for highly sensitive detection. This approach differs from the present invention as the synthetic oligonucleotide is used for subsequent DNA amplification, which is used as the sensitive detection approach.
The use of DNA amplification such as PCR is limited in that it does not permit the use of the synthetic oligonucleotide-antibody conjugate to be used in existing ELISA systems, and it also requires the use of thermal cycling capabilities requiring a dedicated device. Adler describes a related PCR approach to immunoassays in WO2008/122310. A number of related strategies have been reviewed by Adler et al., (2008, “Sensitivity by combination: immuno-PCR and related technologies”, Analyst, vol. 133: 702-718); and Niemeyer et al., (2005, “Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification”, Trends in Biotechnology, vol. 23(4): 208-216).
Cantor and Chuck (U.S. Pat. No. 5,635,602) describe a bis-protein conjugate using avidin/streptavidin binding linkage which they teach for immunoassays and PCR assays. For example, a first antibody is attached by a disulphide bond to DNA and the complimentary DNA at the other end is labeled with biotin. The biotin can bind to streptavidin which also binds biotinylated horseradish peroxidase (HRP). The latter can react with a substrate to generate a chemiluminescent signal. This disclosure differs as it does not teach the use of biotin-streptavidin moieties which do not permit the high degree of control compared to the multivalent streptavidin molecule.
Tennila et al., (2008, “Peptide-oligonucleotide conjugates form stable and selective complexes with Antibody and DNA”, Bioconjugate Chemistry, vol. 19(7): 1361-1367) describe a short oligopeptide conjugated to a synthetic oligonucleotide used for antibody epitope mapping. They are silent on the ELISA assay.
Ketomaki & Lonnberg, 2006, (“A Mixed-Phase Immunoassay Based on Simultaneous Binding of Fluorescently Tagged and PNA-Conjugated peptide Epitopes on Antibodies: Quantification on PNA-Coated Microparticles, Bioconjugate Chemistry, vol. 17:1063-68.) describes a system where a complete antibody (with 2 F(ab) binding moieties) wherein one F(ab) binding moiety binds to a peptide with a fluorescent tag, and the other F(ab) binding moiety binds to a peptide sequence with a nucleic acid sequence (PNA). The PNA binds to the complementary PNA on a microparticle. These authors do not describe using covalent attachment of either the enzyme or the antibody, but rather the use of non-covalent binding of the antibody to a molecule that binds to the microparticle. Another non-covalent binding process is used for the antibody which generates a signal.
Wacker et al., (2004, “Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study”, Analytical Biochemistry, vol. 330:281-287) describe the use of synthetic oligonucleotides covalently attached to capture antibody to generate antibody arrays, which they define as DNA-directed Immobilization (DDI). Another application for synthetic oligonucleotide-antibody conjugates is to immobilize capture antibodies onto solid supports (Jung et al., 2008, “Recent Advances in immobilization methods of antibodies on solid supports”, Analyst, vol. 133: 697-701). This application differs from the present approach in that it does not teach immobilization of antibodies to solid supports. Lovrinovic et al., (2002, “Synthesis of protein-nucleic conjugates by expressed protein ligation”, Chemical Communications, issue 7: 822-823) disclose a protein-nucleic acid conjugate where a recombinant protein is designed with an intein sequence which uses DNA directed immobilization. An intein is an approximately 150 amino acid polypeptide sequence which can excise itself “from a primary translation product with concomitant ligation of the flanking polypeptides (exteins)” (Cook et al., 1995, “Photochemically Initiated protein splicing”, Angewandte Chemie International Edition in English, vol. 34(15):1629-1630) In this paper Lovrinovic used an expressed protein containing a recombinant protein with a covalently bound intein sequence followed by a chitin binding domain. This multifunctional protein hybrid molecule was first purified using the chitin binding domain on a chitin column. Additional molecules containing chemically synthesized cysteine peptides covalently bound to synthetic oligonucleotide sequence are added to the reaction after purification and are bound to the carboxy terminus of the recombinant protein after the excision of the intein region by transesterification followed by ligation of the chemically ligated cysteine-synthetic oligonucleotide hybrid. This now generates a molecule with recombinant protein covalently bound to the cysteine-synthetic oligonucleotide hybrid with no intein sequence. This new molecule can then be used to bind antigen (the recombinant protein) to a complementary synthetic oligonucleotide bound to a solid support, much like DDI technology where the antibody is replaced in this application with an antigen or recombinant protein molecule.
Fan et al., 2008 (“Integrated Barcode chips for rapid, multiplexed analysis of protein in microliter quantities of blood”, Nature Biotechnology, Advance Online Publication) describe yet another variation of this DDI technology.
Heyduk et al., (2008, “Molecular Pincers: Antibody-Based Homogeneous Protein Sensors”, Analytical Chemistry, vol. 80:5152-5159) describes a Fluorescence Resonance Energy Transfer (FRET) based antibody detection technology wherein one antibody molecule with associated first synthetic oligonucleotide which possesses a donor chromophore, and a second antibody molecule which binds to the same antigen at another site on the antigen and which possesses an associated second synthetic oligonucleotide which is complementary to the first synthetic oligonucleotide sequence which possess an acceptor chromophore. The close proximity of the two antibodies on the antigen permit the synthetic oligonucleotide sequences to hybridize and in turn bring the donor and acceptor chromophores in close proximity to each other which reduces the resulting fluorescence. This is a homogeneous antigen detection approach and does not employ a capture antibody.
Niemeyer et al., (2002, “DNA-directed Assembly of Bienzymic complexes from In Vivo Biotinylated NAD(P)H:FMN Oxidoreductase and Luciferase”, (ChemBio Chem, No 02-03: 242-245) describe spatially ordered multienzyme complexes (MECs) using DNA-directed organization, but is silent on the development of ELISA and more sensitive immunoassay tests.
Seeman, (1999, “DNA engineering and its application to nanotechnology”, TIBTECH, vol 17:437-443) describes the application of DNA scaffolding wherein complementary synthetic oligonucleotide sequences can be designed to generate unique structures, but is silent on ELISA assays.
Niemeyer, (2000, “Self-assembled nanostructures based on DNA: towards the development of nanobiotechnology”, Current Opinion in Chemical Biology, vol 4: 609-618) describes the application of DNA to generate scaffolding backbones for ordered structures, but is silent on ELISA and other more sensitive immunoassays.
Storhoff & Mirkin, (1999, “Programmed materials synthesis with DNA”, (Chemical Review, vol. 99: 1849-1862) describes the use of DNA as a scaffolding material but is silent on its use for ELISA assays.
Niemeyer et al., (1998, “Covalent DNA-Streptavidin Conjugates as Building Blocks for novel Biometallic Nanostructures”, Angew. Chem. Int. Ed., vol. 37(16): 2265-2268) describes the use of DNA scaffolds to generate structured molecules with multi-streptavidin molecule aggregates using a biotin streptavidin immunoglobulin attached to this aggregate of biometallic aggregates.
Tomkins et al., (2001, “Preparation of Symmetrical and Unsymmetrical DNA-Protein Conjugates with DNA as a molecular Scaffold”, ChemBio Chem, Issue 5: 375-378), describe generating Streptavidin-DNA conjugates which were imaged by atomic force microscopy for streptavidin-DNA dumb-bells. This reference is silent on ELISA and other sensitive immunoassays.
Takeda et al., (2008, “Covalent split protein fragment-DNA hybrids generated through N-terminus specific modification of proteins by oligonucleotides”, Organic and Biomolecular Chemistry, vol. 6:2187-2194) describe the use of DNA hybrids attached to split proteins to form active enzyme molecules, but do not anticipate ELISA or other sensitive immunoassays.
Niemeyer et al., (1994, “Oligonucleotide-directed self-assembly of proteins: semi-synthetic DNA-streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates”, Nucleic Acids Research, vol. 22(25): 5530-5539) describe a hybrid between an antibody and ALP enzyme using a biotinylated antibody and a biotinylated alkaline phosphatase and streptavidin attached to two DNA sequences wherein the two proteins are bound to each other through biotin-streptavidin binding. This invention describes two molecules scaffolded together directly through DNA hybridization using terminal streptavidin molecules binding to biotinylated antibody and alkaline phosphatase molecules. As the terminal binding moiety is streptavidin, it does not afford the level of controlled synthesis as would be found for covalently bound synthetic oligonucleotide sequences, as for example, the multivalent streptavidin molecule could bind either an antibody or alkaline phosphatase molecule which is biotinylated.
Duckworth et al., (2007, “A Universal Method for the preparation of Covalent Protein-DNA Conjugates for use in Creating Protein Nanostructures”, Angew. Chem. Int. Ed. Vol. 46: 8819-8822) describe the use of DNA-protein structures as scaffolding structures to precisely attach green fluorescent protein, but are silent on ELISA and other immunoassay technology.
Niemeyer, (2000, “Self-assembled nanostructures based on DNA: towards the development of nanobiotechnology”, Current Opinion in Chemical Biology, vol. 4:609-618) describes the use of protein assembled structures using Covalently attached DNA, but is silent on ELISA and other immunoassay technology.
Kawabata et al., (2005, “Liquid-Phase Binding Assay of alpha-Fetoprotein Using DNA-Coupled Antibody and Capillary Chip Electrophoresis”, Analytical Chemistry, vol. 77: 5579-5582) developed a chromatography based immunoassay wherein they added a DNA conjugated antibody molecule with antigen and determined the presence of antigen binding by the increased molecular weight after chromatography, and in this particular assay they used capillary electrophoresis. This method does not use a capture antibody, nor an enzyme-linked conjugate as used in an ELISA assay and therefore differs from the present invention.
Zhang & Guo, (2007, “Multiple labeling of Antibodies with Dye/DNA Conjugate for Sensitivity Improvement in Fluorescence Immunoassay”, Bioconjugate Chemistry, vol. 18(5): 1668-1672) describe a method of immuno-detection that first requires fixing the antigen to a solid support, followed by the addition of a biotinylated antibody, which then uses a biotin-streptavidin-biotin moiety attached to a synthetic oligonucleotide which is fluorescently labeled. This concept differs from the present invention.
Zhu et al. (2008, “Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels”, Analytica Chimica Acta, vol. 624:141-146) used fluorescently tagged DNA to increase signal. The synthetic oligonucleotide contains multiple fluorescent tags and biotin bound to streptavidin, which in turn is bound to biotinylated antibodies. The purpose of this construct was to increase the number of fluorescent tags associated with the antibody and in turn increase the signal level. This differs from the present invention for at least the reason that the synthetic oligonucleotides are covalently attached to the antibody.
Niemeyer et al., (2001, “Nanostructured DNA-Protein Aggregates Consisting of Covalent Oligonucleotide-Streptavidin Conjugates”, Bioconjugate Chemistry, vol. 12(3): 364-371) have generated nanostructures of multimers of streptavidin-biotin-DNA-biotin-streptavidin-biotin-DNA-biotin-streptavidin which binds to biotinylated antibodies to generated structures for use in immuno-PCR reactions. This differs from the present invention.
Kujipers et al., (1993, “Specific Recognition of Antibody-Oligonucleotide Conjugates by Radiolabeled Antisense Nucleotides: A Novel Approach for Two-Step Radioimmunotherapy of Cancer”, Bioconjugate Chemistry, vol. 4: 94-102) generated DNA antibody conjugates which were used for “pre-targeted” radio immunotherapy of cancer patients. This allowed the addition of non-radioactive DNA antibody conjugates to targeted sites, followed by radiolabelled complementary DNA, allowing very specific radiation therapy. The present invention does not deal with radio immunotherapy.